The broad, long-term objective of this proposal is to develop, validate, and employ protein and peptide autoantigen microarrays for profiling autoantibodies found in biological fluids. We will test the hypothesis that large-scale, parallel detection of autoantibody profiles can be used to explore epitope spreading, the role played by a subset of inflammatory cytokines in the initiation and propagation of autoimmunity, and ultimately in selection of antigen-specific tolerizing therapies. We have reduced to practice the use of large-scale arrays to identify autoantibody profiles in many human autoimmune diseases. We will use biochemical, immunological, and molecular biological techniques to validate and extend our ongoing protein array platform in exploring five specific aims in this proposal: (i.) to identify a universal surface chemistry for printing protein, peptide, and ribonucleoprotein complexes; (ii.) to validate autoantibody binding to individual features using highly-characterized serum samples and monoclonal antibodies; (iii.) to construct and validate a comprehensive CTD autoantigen array for detection of autoantibodies; (iv.) to use autoantigen microarrays to characterize serum and tissue-derived autoantibodies from spontaneous and inducible murine models of SLE; and (v.) to test the hypothesis that autoantibody profiling can be used as a surrogate marker of tolerance in animals treated with antigen-specific, DNA plasmid-based, tolerizing vaccines. The results of this proposal may elucidate an expanded role for B lymphocytes and their secreted products in autoimmunity, perhaps heralding an era of customized, antigen- or tissue- specific tolerizing therapy in humans. Moreover, further development of protein microarray technology will have broad applications to the fields of immunology, functional genomics, and proteomics.